Microscopic examination of tissue samples, particularly those obtained by biopsy, is a common method for diagnosis of disease. In particular, immunohistochemistry (IHC), a technique in which specific antibodies are used to detect expression of specific proteins in the tissue sample, may be a valuable tool for diagnosis, particularly for the detection and diagnosis of cancer.
p53 is a tumor suppressor gene that may be mutated in numerous human cancers. (e.g., see article, “p53 Isoforms: An Intracellular Microprocessor?” Khoury M P, Bourdon J C. Genes Cancer. 2011 April; 2(4):453-65, and article “p53/p63/p73 isoforms: an orchestra of isoforms to harmonise cell differentiation and response to stress.” Murray-Zmijewski F, Lane D P, Bourdon J C. Cell Death Differ. 2006 June; 13(6):962-72, each hereby incorporated by reference herein.) Inactivation of p53 may be one of the most common genetic alterations in human cancers and may even be present in about 50% of all human cancers. p53 may respond to a wide variety of cellular stresses, including DNA damage, hypoxia, and metabolic changes, perhaps by activating cellular pathways that may result in cell cycle arrest or apoptosis. p73 may have been cloned and identified as a member of the p53 family, based on sequence homology with key regions of p53. p73 may have demonstrated similar transcriptional activities to p53; however, p73 may not exhibit different activities than p53. (e.g., see article, “Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers.” Kaghad M, Bonnet H, Yang A, Creancier L, Biscan J C, Valent A, Minty A, Chalon P, Lelias J M, Dumont X, Ferrara P, McKeon F, Caput D. Cell. 1997 Aug. 22; 90(4):809-19, each hereby incorporated by reference herein.)
Using PCR primers based on the DNA binding domains of p53 and p73, a gene may have been cloned and the cloned genetic sequence may even have corresponded to a protein of approximately 40-kDa. Therefore, this newly identified gene, an additional member of the p53 family may have been identified as p40. (e.g., see article, “A new human p53 homologue.” Trink B, Okami K, Wu L, Sriuranpong V, Jen J, Sidransky D. Nat Med. 1998 July; 4(7):747-8, hereby incorporated by reference herein.) Other isoforms of the same gene may have been cloned and their products may have been referred to as p51, p63, or p73L. (e.g., see articles, “Cloning and functional analysis of human p51, which structurally and functionally resembles p53.” Osada M, Ohba M, Kawahara C, Ishioka C, Kanamaru R, Katoh I, Ikawa Y, Nimura Y, Nakagawara A, Obinata M, Ikawa S. Nat Med. 1998 July; 4(7):839-43, and article, “p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.” Yang A, Kaghad M, Wang Y, Gillett E, Fleming M D, Dötsch V, Andrews N C, Caput D, McKeon F. Mol Cell. 1998 September; 2(3):305-16, and article, “A second p53-related protein, p73L, with high homology to p73.” Senoo M, Seki N, Ohira M, Sugano S, Watanabe M, Inuzuka S, Okamoto T, Tachibana M, Tanaka T, Shinkai Y, Kato H. Biochem Biophys Res Commun. 1998 Jul. 30; 248(3):603-7, and article, “Cloning and functional analysis of human p51, which structurally and functionally resembles p53.” Osada M, Ohba M, Kawahara C, Ishioka C, Kanamaru R, Katoh I, Ikawa Y, Nimura Y, Nakagawara A, Obinata M, Ikawa S. Nat Med. 1998 July; 4(7):839-43, each hereby incorporated by reference herein.) Each of these isoforms may differ by the presence or even absence of an N-terminal transcriptional activating domain. p40, ΔNp63 and p73L may lack this domain.
The p40/p63/p51/p73L gene may have been found to be overexpressed in cell lines of head and neck squamous cell carcinomas and primary lung squamous cell carcinomas. (e.g., see article, “AIS is an oncogene amplified in squamous cell carcinoma.” Hibi K, Trink B, Patturajan M, Westra W H, Caballero O L, Hill D E, Ratovitski E A, Jen J, Sidransky D. Proc Natl Acad Sci USA. 2000 May 9; 97(10):5462-7, hereby incorporated by reference herein.) Due to this amplification in squamous cell carcinomas, the p40/p63/p51/p73L gene may be referred to as AIS (amplified in squamous cell carcinoma). The longest isoforms of the p40/p63/p51/p73L gene, perhaps including transcriptional activation domains, may be those cloned and named as p63. (e.g., see article, “p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.” Yang A, Kaghad M, Wang Y, Gillett E, Fleming M D, Dötsch V, Andrews N C, Caput D, McKeon F. Mol Cell. 1998 September; 2(3):305-16, hereby incorporated by reference herein.)
Therefore, for simplicity, the p40/p63/p51/p73L gene may frequently be referred to as p63; however, it should be noted that p40, p63, p51, p73L, and AIS are all terms for the same gene, which may produce transcripts of various lengths, perhaps resulting in protein isoforms of correspondingly various lengths.
The full length of the p63 gene may encode an N-terminal transcriptional activation domain, a DNA binding domain, and a carboxy-oligomerization domain. p63 may have two promoters, which perhaps results in two distinctly different classes of proteins, where one class may contain the transactivation domain (TA) (perhaps known as p63 or TAp63) and the other class may lack the N-terminal transactivation domain (perhaps known as p40 or ANp63). (e.g., see article, “p40: A p63 Isoform Useful for Lung Cancer Diagnosis—A Review of the Physiological and Pathological Role of p63.” Nobre A R, Albergaria A, Schmitt F. Acta Cytol. 2013; 57(1):1-8, hereby incorporated by reference herein.) The p40 protein may act as a dominant negative factor for transcription by either competing for DNA binding sites or directly binding to p53, p63, or p73 isoforms, perhaps inhibiting their ability to induce transcription. However, in some instances, p40 may activate transcription of genes that are perhaps otherwise not activated by the TA forms of p53, p63, or p73. In one view, the p63 gene may be thought of as producing a family of opposing molecules: perhaps proteins containing a N-terminal transactivation domain, with p53-like tumor suppressor properties, and perhaps proteins lacking an N-terminal domain, with oncogenic properties.
In normal tissue of adults, expression of p63 isoforms may be restricted to the nuclei of basal cells of normal epithelia, such as skin, esophagus, and urothelium or the like, and basal cells of glandular structures in prostate, breast and bronchi or the like. In these cells, expression of p40 may be found to be perhaps about 100-fold higher than that of TAp63. Although p40 and TAp63 may exhibit overlapping tissue distributions, TAp63 may be more expressed in differentiated cells and perhaps less expressed in basal cells, relative to p40 expression. Differences in expression patterns for the isoforms may perhaps indicate different roles for each isoform in development and disease. (e.g., see article, “p40: A p63 Isoform Useful for Lung Cancer Diagnosis—A Review of the Physiological and Pathological Role of p63.” Nobre A R, Albergaria A, Schmitt F. Acta Cytol. 2013; 57(1):1-8, hereby incorporated by reference herein.)
Determining expression levels of the TAp63 and p40 isoforms may have been useful in diagnosis of cancer. In particular, IHC, using antibodies that bind the TAp63 and/or p40 proteins may have been useful for detecting protein expression and perhaps diagnosing cancer, particularly lung, prostate, breast, and bladder cancer. A rabbit polyclonal (RP) anti-p40 antibody may have been produced and used in an IHC method to identify expression of p40 in head and neck squamous cell carcinoma cell lines and primary lung tumors of the squamous cell carcinoma type. (e.g., see article, “A new human p53 homologue.” Trink B, Okami K, Wu L, Sriuranpong V, Jen J, Sidransky D. Nat Med. 1998 July; 4(7):747-8, hereby incorporated by reference herein.) A mouse monoclonal anti-p63 antibody (4A4) may also have been produced and used in an IHC method to detect isoforms of p63 protein. (e.g., see article, “p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.” Yang A, Kaghad M, Wang Y, Gillett E, Fleming M D, Dötsch V, Andrews N C, Caput D, McKeon F. Mol Cell. 1998 September; 2(3):305-16, hereby incorporated by reference herein.) Importantly, the RP anti-p40 antibody may bind an epitope sequence that is unique to the p40 isoform, a sequence that is perhaps not present in TAp63. In contrast, the anti-p63 antibody 4A4 may bind an epitope sequence that may be common to both the TA and p40 isoforms, perhaps resulting in 4A4 exhibiting properties of a pan-p63 marker by recognizing both classes of proteins derived from the p63 gene, whereas the RP anti-p40 antibody may recognize only the p40 isoform and may perhaps be a more selective marker.
Lung cancer is the leading cause of cancer death for both men and women. More people die of lung cancer than of colon, breast, and prostate cancers combined. Non-small cell lung carcinoma (NSCLC) comprises approximately 80% of lung cancers and may be classified into several histological types, most commonly may include adenocarcinoma (ADC) or even squamous cell carcinoma (SCC). Classification of lung carcinomas into histological types may be performed by morphological examination using hematoxylin and eosin (H&E) or IHC, and in some cases even, mucin stains; however, accurate classification can be difficult with poorly differentiated or even undifferentiated lung carcinoma. Diagnosis can be further complicated by the use of needle core biopsies, which may provide limited amounts of tissue for both immunohistochemistry and molecular testing, and may include crush artifacts. Additionally, cytology specimens may lack morphological features necessary for diagnosis with H&E alone.
Although the majority of lung cancers (particularly grades I and II) can be diagnosed with only H&E staining, with the advent of targeted therapies, diagnostic needs have changed, and an improved method for classification of a greater number of NSCLC cases is needed. In the past, histologic subtyping of NSCLCs had limited diagnostic value, due to the fact that the same treatment may have been provided to the patient, perhaps regardless of NSCLC subtype. However, the availability of targeted therapies has created a need for accurate subtyping of NSCLC. For example, bevacizumab, a therapeutic humanized monoclonal antibody targeting vascular endothelial growth factor, may be a common treatment for NSCLC patients; however, patients with the SCC subtype should not receive bevacizumab, perhaps due to about 30% mortality rate by fatal pulmonary hemorrhage. Furthermore, enhanced efficacy may have been demonstrated with the addition of premextred to conventional chemotherapy in non-squamous cell carcinomas, but may not in SCC. Therefore, accurate methods for subtyping NSCLC specimens may be useful for the best patient care, with optimal therapeutic efficacy and minimal adverse effects.
IHC may be commonly used to assist pathologists in determining histologic subtype of NSCLC specimens, perhaps particularly discriminating ADC from SCC, as well as from Small Cell Carcinomas of the lung. In particular, IHC antibodies to p40 may be useful in the diagnosis of lung cancer, and perhaps in discriminating histologic subtypes, such as ADC and SCC. Using the RP anti-p40 antibody, expression of p40 protein in primary lung SqCC may have been identified by IHC; whereas, p40 protein may not have been detected in cases of lung adenocarcinoma. (e.g., see article, “AIS is an oncogene amplified in squamous cell carcinoma.” Hibi K, Trink B, Patturajan M, Westra W H, Caballero O L, Hill D E, Ratovitski E A, Jen J, Sidransky D. Proc Natl Acad Sci USA. 2000 May 9; 97(10):5462-7, hereby incorporated by reference herein.)
Further studies may have shown that the RP anti-p40 antibody may be superior to the pan-p63 antibody 4A4 for the diagnosis of squamous cell carcinoma, and that perhaps the RP anti-p40 antibody may be more specific. In one study, both the RP anti-p40 antibody and pan-p63 antibody stained all 81 cases of SCC that were evaluated, perhaps indicating that both antibodies exhibit equal sensitivity in SCC. In contrast, the pan-p63 antibody stained 74/237 (about 31%) of ADC cases, while the RP anti-p40 antibody stained only 7/205 (about 3%) of ADC cases, perhaps indicating that the RP anti-p40 antibody may be more specific than the pan-p63 4A4. Furthermore, the pan-p63 4A4 antibody stained 82/152 (about 54%) of the large cell lymphoma cases tested, whereas, the RP anti-p40 antibody lacked staining in any of these cases, perhaps resulting in improved specificity for the RP anti-p40 antibody. This study may have shown that IHC detection of p40 instead of detection of multiple p63 isoforms with 4A4 prevents misinterpretation of poorly differentiated ADC or lymphoma as SCC. (e.g., see article, “p40 (ΔNp63) is superior to p63 for the diagnosis of pulmonary squamous cell carcinoma.” Bishop J A, Teruya-Feldstein J, Westra W H, Pelosi G, Travis W D, Rekhtman N. Mod Pathol. 2012 March; 25(3):405-15 and article, “A study of ΔNp63 expression in lung non-small cell carcinomas.” Nonaka D. Am J Surg Pathol. 2012 June; 36(6):895-9, each hereby incorporated by reference herein.)
Other studies may also have reported use of the RP anti-p40 antibody in identifying SCC, perhaps in combination with an anti-TTF-1 antibody, which may be a marker for ADC. In one such study, using IHC of small tissue samples from biopsy specimens, 45/46 (about 98%) cases of ADC and SCC were diagnosed, using a combination of RP anti-p40 and anti-TTF-1 antibodies (e.g., see article, “ΔNp63 (p40) and thyroid transcription factor-1 immunoreactivity on small biopsies or cellblocks for typing non-small cell lung cancer: a novel two-hit, sparing-material approach.” Pelosi G, Fabbri A, Bianchi F, Maisonneuve P, Rossi G, Barbareschi M, Graziano P, Cavazza A, Rekhtman N, Pastorino U, Scanagatta P, Papotti M. J Thorac Oncol. 2012 February; 7(2):281-90, hereby incorporated by reference herein.)
In a similar study, an antibody cocktail containing the RP anti-p40 antibody and an anti-TTF-1 antibody resulted in about 92% sensitivity and about 93% specificity of the RP anti-p40 antibody for SCC, and about 77% sensitivity and about 100% specificity of anti-TTF-1 for ADC. (e.g., see article, “Tissue-Preserving Antibody Cocktails to Differentiate Primary Squamous Cell Carcinoma, Adenocarcinoma, and Small Cell Carcinoma of Lung.” Brown A F, Sirohi D, Fukuoka J, Cagle P, Policarpio-Nicolas M, Tacha D, Jagirdar J. Arch Pathol Lab Med. 2013 Jan. 4, hereby incorporated by reference herein.)
In normal bladder tissue, TAp63 may be expressed in basal and intermediate urothelial cells, whereas p40 may not be detected. In contrast, p40 expression may be detected by IHC in urothelial carcinoma, perhaps in 29/147 (about 19.7%) of non-invasive cases, and possibly 23/55 (about 41.8%) cases of invasive carcinoma. (e.g., see article, “Distinct expression profiles of p63 variants during urothelial development and bladder cancer progression.” Karni-Schmidt O, Castillo-Martin M, Shen T H, Gladoun N, Domingo-Domenech J, Sanchez-Carbayo M, Li Y, Lowe S, Prives C, Cordon-Cardo C. Am J Pathol. 2011 March; 178(3):1350-60, hereby incorporated by reference herein.) Expression of p40 may also be a prognostic factor in urothelial carcinoma. For example, cases of muscle invasive urothelial carcinoma that demonstrate expression of p40 may exhibit a median±SD overall survival of perhaps about 11.6±about 1.3 months, perhaps indicating that p40 expression is a predictor of poor prognosis. In contrast, cases of invasive urothelial carcinoma that lack p40 expression may have demonstrated a longer median±SD overall survival of perhaps about 25±about 6.4 months, perhaps indicating that a lack of p40 expression is a predictor of better prognosis. Notably, total p63 expression may not be a prognostic indicator in invasive carcinoma, perhaps because TAp63 and p40 isoforms exhibit opposing biological activities. As a result, a marker that is specific for the p40 isoform may be used for urothelial carcinoma, compared to a pan-p63 marker.
Loss of expression of p40, which may normally be expressed in basal cells of prostate glands, may commonly be used as an indicator adenocarcinoma of the prostate. Specifically, the quantity of p40 mRNA transcript may be lower, perhaps approximately about 2000-times lower or perhaps even undetectable, in prostate cancer cell lines compared to the amount of TAp63 mRNA. Therefore, perhaps p40 is virtually absent in prostate cancer cell lines, whereas TAp63 mRNA is detectable at various levels. In the differential diagnosis of benign and malignant prostate tissue, a method that may detect the loss of p40 may be preferred over a method that may detect TAp63 (e.g., see article, “p63 is a prostate basal cell marker and is required for prostate development.” Signoretti S, Waltregny D, Dilks J, Isaac B, Lin D, Garraway L, Yang A, Montironi R, McKeon F, Loda M. Am J Pathol. 2000 December; 157(6):1769-75, and article, “Differential expression of p63 isoforms in normal tissues and neoplastic cells.” Nylander K, Vojtesek B, Nenutil R, Lindgren B, Roos G, Zhanxiang W, Sjöström B, Dahlqvist A, Coates P J. J Pathol. 2002 December; 198(4):417-27, each hereby incorporated by reference herein.)
In normal breast ducts, myoepithelial cells may form a continuous basal rim along the epithelial structure. The gradual loss of continuity of this basal boundary may be indicative of carcinoma in situ, and the total loss of myoepithelial cells may often be observed in invasive breast carcinoma. Benign lesions may typically retain their continuous myoepithelial layer, while morphologically resembling a malignant lesion. Nuclear staining of myoepithelial cells by IHC may be observed using an anti-p40 antibody. In this manner, an anti-p40 antibody may be useful for evaluating the continuity of the myoepithelial cells in the basal layer and thus aid in the diagnosis of breast lesions. (e.g., see article, “p63, a p53 homologue, is a selective nuclear marker of myoepithelial cells of the human breast.” Barbareschi M, Pecciarini L, Cangi M G, Macrì E, Rizzo A, Viale G, Doglioni C. Am J Surg Pathol. 2001 August; 25(8):1054-60, hereby incorporated by reference herein.)
Therefore, a clear need exists for a sensitive and even specific anti-p40 antibody for use in cancer diagnosis. Embodiments of the present invention provide an anti-p40 mouse monoclonal antibody [clone BC28] which may be highly sensitive and may even be highly specific. An example of the present invention provides a mouse monoclonal anti-p40 antibody that may detect the presence or absence of p40 protein in certain cancers, including but not limited to SCC, bladder, breast and prostate cancer or the like. In cases of NSCLC, an example of the present invention may have demonstrated excellent sensitivity for lung SCC (about 65/67, about 97%) with perhaps even excellent specificity versus lung ADC (about 0/71, about 0%). When compared to the RP anti-p40 antibody, the mouse monoclonal anti-p40 BC28 may have typically demonstrated cleaner staining patterns, perhaps with fewer artifacts, as well as a lack of staining of macrophages, while even offering the advantages of a monoclonal antibody. BC28 also may not stain some specimens of lung ADC, which may have been stained by the RP anti-p40 antibody, perhaps indicating the superior specificity of BC28 over alternatives. Therefore, a monoclonal anti-p40 antibody, such as BC28, may be preferred for diagnosis, compared to other pan-p63 markers or alternative anti-p40 antibodies.
The development of an anti-p40 antibody may aid in the diagnosis of primary and even metastatic cancers, particularly lung SCC, urothelial carcinoma, prostate adenocarcinoma, and breast carcinoma or the like, and may even aid in distinguishing protein expression of p40 versus TAp63. New anti-p40 antibodies such as mouse monoclonal anti-p40 antibody [BC28], with perhaps equal or superior staining sensitivity, and perhaps even superior staining specificity such as compared to the pan-p63 marker 4A4 and the RP anti-p40 antibody, have been provided in the present invention.